The proposed studies will investigate non-conventional roles for voltage-gated ion channels in nervous system development. While voltage-gated sodium channels are well known for their roles in regulating events that occur on a millisecond time scale, such action potential generation, recent studies suggest they have much longer lasting effects on developmental processes. Previous studies demonstrate that the voltage-gated sodium channel navl .6a regulates development of ventrally-projecting secondary motor axons in zebrafish embryos. Upon knock-down of navl .6a using antisense morpholinos that bind scnSaa mRNA (which encodes the nav1.6a protein) and prevent translation of nav1.6a protein, ventrally-projecting motor axons appear branched and defasciculated and often turn before reaching their ventral targets. Interestingly, expression studies suggest that scnSaa mRNA is not expressed in ventrallyprojecting secondary motor neurons, the cells affected by loss of navl .6a. Mosaic embryos in which navl .6a is knocked down in individual cells or small groups of cells in the embryo confirmed that motor axons develop abnormally when navl .6a is knocked down in spinal cord cells other than the secondary motor neurons themselves. These results demonstrate non-cell autonomous effects of navl .6a on nervous system development, suggesting new roles for voltage-gated sodium channels. The overall goal of the proposed study is to investigate the role of navl .6a in development of ventrally-projecting secondary motor neuron axons. The three specific aims of this study are: (l)determine when defects in ventrally-projecting axons of secondary motor neurons first appear;(2)identify scnSaa-expressing cells in the developing zebrafish spinal cord;and (3)test the hypothesis that the scnSaa-expressing cells mediating effects on ventral motor axon branching and fasciculation are glia and/or spinal interneurons. The study proposes to use timelapse microscopy to determine when defects in ventral motor axons first become apparent upon knock-down of navl .6a by antisense morpholinos. In situ hybridization and immunocytochemistry will colocalize scnSaa mRNA with markers of identified spinal interneurons and glia to identify the spinal cord cells that express scnSaa mRNA during development. Furthermore, generation of embryos with mosaic embryos will be used to determine in which cells navl .6a acts to regulate ventral motor axon development. Relevance: Mice lacking Navl .6a develop dystonias and neurodegeneration, similar to human dystonias. The results of these studies have the potential to further our understanding of disease mechanisms involving Nav1.6.